IL-21 Derivatives and variants

ABSTRACT

The invention provides derivatives of IL-21 or variants thereof, methods of producing such variants, new variants of IL-21, and various methods of using such molecules.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of copending InternationalPatent Application No. PCT/DK2004/000686 (published as WO 2005/035565),filed Oct. 8, 2004 (which designates the US) and claims the benefit(under 35 USC §119) of U.S. Provisional Patent Applications 60/510,892,60/513,422, and 60/569,566, filed Oct. 14, 2003, Oct. 22, 2003, and May10, 2004, respectively, and Danish Patent Applications PA 2003 01496, PA2003 01529, and PA 2004 00707, filed Oct. 10, 2003, Oct. 17, 2003, andMay 4, 2004, respectively, the entirely of each of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to derivatives of interleukin 21 (IL-21) and IL-21variants, as well as the synthesis and purification of derivatives ofIL-21 and analogues thereof (including variants of IL-21 which act asIL-21 antagonists).

BACKGROUND OF THE INVENTION

IL-21 was described for example in WO 00/53761 as a stimulator of T cellgrowth and NK activity. Derivatives of IL-21 have previously not beendescribed. As a potent pharmaceutical derivatives with modifiedcharacteristics are interesting in many applications.

SUMMARY OF THE INVENTION

The invention provides derivatives of IL-21 or variants thereof.

In an aspect the invention provides derivatives of IL-21 or variantsthereof which comprises a polymeric molecule or lipophilic derivative(substituent).

In an aspect of the invention the derivative of IL-21 or variantsthereof, comprises a polymeric molecule which is one or more PEG groups.

In an aspect of the invention derivatives of IL-21 or variants thereof,comprises derivatisation in the N-terminal, or the C-terminal orinternally in the molecule.

In an aspect of the invention the derivatisation is on a naturallyoccuring amino acid, and/or also or alternatively one an aminoacid addedor substituted into the IL-21 sequence.

The invention provides the specific variant Ser-hIL21, isolated DNAexpressing the specific variant and the use for derivatisation with apolymeric molecule. The invention also provides the use of thederivatives of IL-21 or variants thereof, for the manufacture of amedicament for the treatment of cancer of infectious diseases.

DESCRIPTION OF THE INVENTION

The invention provides various derivatives of the IL-21 peptides. Thederivatives include chemically modified peptides that comprise an IL-21peptide, or variants of the IL-21 peptide. Chemical modification mayalter the chemical and biological characteristics of a moleculedependent on the characteristics of the derivatising molecule. Theeffect of modification may be maintaining the biological function of thepeptide or potentially a lower activity of the peptide. For examplederivatisation may extend the functional in vivo half life of aderivatised peptide and thus compensate for a lower activity. Forexample a protracted profile effect of IL-21 derivatives may be achievedby coupling of a IL-21 peptide or an analogue thereof to a hydrophilicmoiety that results in IL-21 derivatives with a maintained biologicalactivity. The derivatisation may for example provide a peptide with animproved half-life, thereby facilitating the continuous presence oftherapeutically effective amount of IL-21 or a derivative thereof havingthe same biological effect. The amount needed for administration of aneffective amount of a protracted peptide may thus be lower.Derivatisation may protect the molecule against degradation by enzymesand prevent clearence from the body. The derivatisation is preferablynon-immugenic. In an aspect of the invention the solubility of thepeptide may be amended.

IL-21 activity is as defined as described in Parrish-Novak, Nature, 408,57-63, 2000; Brady, J., Hayakawa, Y., Smyth, M. J., and Nutt, S. L.2004. IL-21 induces the functional maturation of murine NK cells.Journal of immunology (Baltimore, Md. 172:2048-2058; Collins, M.,Whitters, M. J., and Young, D. A. 2003. IL-21 and IL-21 receptor: a newcytokine pathway modulates innate and adaptive immunity. Immunol Res28:131-140; Habib, T., Nelson, A., and Kaushansky, K. 2003. IL-21: anovel IL-2-family lymphokine that modulates B, T, and natural killercell responses. J Allergy Clin Immunol 112:1033-1045. Sivakumar, P. V.2004. Interleukin-21 is a T-helper cytokine that regulates humoralimmunity and cell-mediated anti-tumour responses. Immunology 112:177;Wang, G., Tschoi, M., Spolski, R., Lou, Y., Ozaki, K., Feng, C., Kim,G., Leonard, W. J., and Hwu, P. 2003. In vivo antitumor activity ofinterleukin 21 mediated by natural killer cells. Cancer Res63:9016-9022; Wang, G. 2003. In vivo antitumor activity of interleukin21 mediated by natural killer cells. Cancer Res 63:9016. IL-21 andderivatives thereof are considered useful in the treatment of neoplasticdisorders. Neoplastic disorders or cancer are to be understood asreferring to all forms of neoplastic cell growth, including both cysticand solid tumors, bone and soft tissue tumors, including both benign andmalignant tumors, including tumors in anal tissue, bile duct, bladder,blood cells, bone, bone (secondary), bowel (colon & rectum), brain,brain (secondary), breast, breast (secondary), carcinoid, cervix,children's cancers, eye, gullet (oesophagus), head & neck, kaposi'ssarcoma, kidney, larynx, leukaemia (acute lymphoblastic), leukaemia(acute myeloid), leukaemia (chronic lymphocytic), leukaemia (chronicmyeloid), leukaemia (other), liver, liver (secondary), lung, lung(secondary), lymph nodes (secondary), lymphoma (hodgkin's), lymphoma(non-hodgkin's), melanoma, mesothelioma, myeloma, ovary, pancreas,penis, prostate, skin, soft tissue sarcomas, stomach, testes, thyroid,unknown primary tumor, vagina, vulva, womb (uterus). Soft tissue tumorsinclude Benign schwannoma Monosomy, Desmoid tumor, Lipo-blastoma,Lipoma, Uterine leiomyoma, Clear cell sarcoma, Dermatofibrosarcoma,Ewing sarcoma, Extraskeletal myxoid chondrosarcoma, Liposarcoma myxoid,Liposarcoma, well differentiated, Alveolar rhabdomyosarcoma, andSynovial sarcoma. Specific bone tumor include Nonossifying Fibroma,Unicameral bone cyst, Enchon-droma, Aneurysmal bone cyst, Osteoblastoma,Chondroblastoma, Chondromyxofibroma, Ossifying fibroma and Adamantinoma,Giant cell tumor, Fibrous dysplasia, Ewing's Sarcoma, EosinophilicGranuloma, Osteosarcoma, Chondroma, Chondrosarcoma, Malignant FibrousHistiocytoma, and Metastatic Carcinoma. Leukaemias referes to cancers ofthe white blood cells which are produced by the bone marrow. Thisincludes but are not limited to the four main types of leukaemia; acutelymphoblastic (ALL), acute myeloblastic (AML), chronic lymphocytic (CLL)and chronic myeloid (CML).

Prior to a discussion of the detailed embodiments of the invention, adefinition of specific terms related to the main aspects of theinvention is provided.

In the context of the present invention IL-21 is defined as the sequencedisclosed in WO00/53761 as SEQ ID No.:2. or the same sequence withoutthe N-terminal sequence. The present application also describes variantsand derivatives of IL-21. In the context of the present invention theterm “IL-21” thus means IL-21as described in WO00/53761 optionallywithout the N-terminal sequence. The present invention embracescounterpart proteins and from other species (“orthologs”). Of particularinterest are IL-21 polypeptides from other mammalian species, includingrodent, porcine, ovine, bovine, canine, feline, equine, and otherprimates.

“IL-21 derivatives” comprises derivatisation or linking to anotherfunctional molecule. The linking can be chemical coupling, geneticfusion, non-covalent association or the like, to other molecularentities such as antibodies, toxins, radioisotope, cytotoxic orcytostatic agents or polymeric molecules or lipophilic groups.Non-limiting examples include polymeric groups such as, e.g, dendrimersas disclosed in PCT/DK2004/000531, polyalkylene oxide (PAO),polyalkylene glycol (PAG), polyethylene glycol (PEG), polypropyleneglycol (PPG), branched PEGs, polyvinyl alcohol (PVA), polycarboxylate,poly-vinylpyrolidone, polyethylene-co-maleic acid anhydride,polystyrene-co-maleic acid anhydride, dextran, carboxymethyl-dextran;serum protein binding-ligands, such as compounds which bind to albumin,like fatty acids, C₅-C₂₄ fatty acid, aliphatic diacid (e.g. C₅-C₂₄).Albumin binders are described in Danish patent applicationsPCT/DK04/000625. Albumin binders are also compounds of the followingformula:

Other examples of protracting groups includes small organic moleculescontaining moieties that under physiological conditions alters chargeproperties, such as carboxylic acids or amines, or neutral substituentsthat prevent glycan specific recognition such as smaller alkylsubstituents (e.g., C₁-C₅ alkyl).

Variants or analogues of IL-21 peptides are characterized as having oneor more amino acid substitutions, deletions or additions. These changesare typically of a minor nature, that is conservative amino acidsubstitutions and other substitutions that do not significantly affectthe receptor binding, receptor affinity, folding or biological activityof the peptide; However, as described below even small amendments inessential aminoacids changes the effect of the IL-21 peptide. Smalldeletions, typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or asmall extension that facilitates purification (an affinity tag), such asa poly-histidine tract, protein A, Nilsson et al., EMBO J. 4:1075(1985); Nilsson et al., Methods Enzymol. 198:3 (1991), glutathione Stransferase, Smith and Johnson, Gene 67:31 (1988), or other antigenicepitope or binding domain. See, in general Ford et al., ProteinExpression and Purification 2: 95-107 (1991). DNAs encoding affinitytags are available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.). Variants of IL-21 peptides may also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,Nmethylglycine, addo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating nonnaturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Essentialamino acids in the polypeptides of the present invention can beidentified according to procedures known in the art, such assite-directed mutagenesis or alaninescanning mutagenesis [Cunningham andWells, Science 244: 1081-1085 (1989)]; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-4502 (1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.,ligand binding and signal transduction) to identify amino acid residuesthat are critical to the activity of the molecule. Sites ofligand-protein interaction can also be determined by analysis of crystalstructure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. The identities ofessential amino acids can also be inferred from analysis of homologieswith related proteins.

In one embodiment a variant is 70% or more identical with the sequenceof SEQ ID NO:2 of WO0053761. In one embodiment a variant is 80% or moreidentical with the SEQ ID NO:2 of WO0053761. In another embodiment avariant is 90% or more identical with the sequence of SEQ ID NO:2 ofWO0053761. In a further embodiment a variant is 95% or more identicalwith the sequence of SEQ ID NO:2 of WO0053761.

Percentage sequence identity between two amino acid sequences isdetermined by a Needelman-Wunsch alignment, useful for both protein andDNA alignments. For protein alignments the default scoring matrix usedis BLOSUM50, and the penalty for the first residue in a gap is −12,while the penalty for additional residues in a gap is −2. The alignmentmay be made with the Align software from the FASTA package version v20u6(W. R. Pearson and D. J. Lipman (1988), “Improved Tools for BiologicalSequence Analysis”, PNAS 85:2444-2448; and W. R. Pearson (1990) “Rapidand Sensitive Sequence Comparison with FASTP and FASTA”, Methods inEnzymology, 183:63-98).

Non-limiting examples of IL-21 variants having substantially modifiedbiological activity relative to wild-type IL-21 is described inWO03/40313 wherein substitutions of single amino acids in the IL-21sequence antagonises the effect of IL-21.

According to this invention antagonists of IL-21 compounds which inhibitthe activity normally observed with IL-21. Such compounds may be as wellsmall molecules, peptides or soluble receptors interacting with IL-21.According to the present invention derivatisation of the antagonists ofIL-21 are peptides or soluble receptors. In an aspect of the inventionthe peptides are IL-21 analogues or variants having an antagonisticeffect.

Antagonists are also fusion protein that includes the extracellulardomain of the IL-21 R fused to an Fc immunoglobulin region, Examples ofantagonistic fusion proteins are shown in SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ II) NO:31, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:37, or SEQ ID NO:39, of WO03/28630.

Other IL-21 antagonists to be used according to the invention are thesequences 4 and 6 of WO03/40313. Tthe IL-21 peptides with variations inone or both of the positions 114 and 119 as mentioned in WO03/87320.

Soluble receptors of IL-21 having antagonistic effect on IL-21 aredisclosed in WO04/07682.

Examples of antagonistic fusion proteins that can be used in the methodsof the invention are shown in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37,and SEQ ID NO:39, of WO03/28630.

Other IL-21 antagonists that can be used in inventive methods providedhere are SEQ ID NOS 4 and 6 of WO03/40313 and IL-21 peptides withvariations in one or both of the positions 114 and 119 of human IL-21 asmentioned in WO03/87320. WO04/07682 describes soluble receptors havingantagonistic activity against IL-21.

In an aspect of the invention, IL-21 antibodies are used as IL-21antagonists. Such antibodies can be produced by any suitable methodknown in the art and examples of such antibodies are described inWO00/53761. IL-21 antagonist antibodies are characterised by inhibitingone or more biological activities of IL-21. Inhibition of biologicactivity can be measured by, e.g., the Ba F3 assay where Ba F3 cellsstably transfected with human IL-21 R (IL-21 R-Ba F3) undergoproliferation when IL-21 is added to the culture. Addition of IL-21antagonists to the IL-21 R-Ba F3 cells desirably partially or fullyinhibits IL-21-dependent proliferation of IL-21 R-Ba F3 cells.

The term “polymeric molecule”, or “polymeric group” or “polymericmoiety” or “polymer molecule”, encompasses molecules formed by covalentlinkage of two or more monomers wherein none of the monomers is an aminoacid residue. Preferred polymers are polymer molecules selected from thegroup consisting of dendrimers as disclosed in PCT/DK2004/000531,polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such aspolyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs,polyvinyl alcohol (PVA), polycarboxylate, poly-vinylpyrolidone,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, and dextran, including carboxymethyl-dextran, PEG beingparticularly preferred.

The term “PEGylated IL-21” means IL-21, having one or more PEG moleculeconjugated to a human IL-21 polypeptide. It is to be understood, thatthe PEG molecule may be attached to any part of the IL-21 polypeptideincluding any amino acid residue or carbohydrate moiety of the IL-21polypeptide. The term “cysteine-PEGylated IL-21” means IL-21 having aPEG molecule conjugated to a sulfhydryl group of a cysteine introducedin IL-21.

The term “polyethylene glycol” or “PEG” means a polyethylene glycolcompound or a derivative thereof, with or without coupling agents,coupling or activating moeities (e.g., with thiol, triflate, tresylate,azirdine, oxirane, or preferably with a maleimide moiety). Compoundssuch as maleimido monomethoxy PEG are exemplary of activated PEGcompounds of the invention. The term “PEG” is intended to indicatepolyethylene glycol of a molecular weight between 500 and 150,000 Da,including analogues thereof, wherein for instance the terminal OH-grouphas been replaced by a methoxy group (referred to as mPEG).

In the present context, the words “peptide” and “polypeptide” and“protein” are used interchangeably and are intended to indicate thesame.

In the context of the present invention “treatment” or “treating” refersto preventing, alleviating, managing, curing or reducing the diseasee.g. a symptom of he disease, a condition underlying the disease orboth.

The term “functional in vivo half-life” is used in its normal meaning,i.e., the time at which 50% of the biological activity of thepolypeptide or conjugate is still present in the body/target organ, orthe time at which the activity of the polypeptide or conjugate is 50% ofits initial value. As an alternative to determining functional in vivohalf-life, “serum half-life” may be determined, i.e., the time at which50% of the polypeptide or conjugate molecules circulate in the plasma orbloodstream prior to being cleared. Determination of serum-half-life isoften more simple than determining functional half-life and themagnitude of serum-half-life is usually a good indication of themagnitude of functional in vivo half-life. Alternative terms to serumhalf-life include plasma half-life, circulating half-life, circulatoryhalf-life, serum clearance, plasma clearance, and clearance half-life.The functionality to be retained is normally selected from procoagulant,proteolytic, co-factor binding, receptor binding activity, or other typeof biological activity associated with the particular protein.

The term “increased” with respect to the functional in vivo half-life orplasma half-life is used to indicate that the relevant half-life of thepolypeptide or conjugate is statistically significantly increasedrelative to that of a reference molecule, such as non-conjugatedglycoprotein as determined under comparable conditions. For instance therelevant half-life may be increased by at least about 25%, such as by atleast about 50%, e.g., by at least about 100%, 150%, 200%, 250%, or500%.

In one embodiment the present invention relates to a use of a derivativeof IL-21 for the preparation of a medicament for the treatment ofdiseases responsive to stimulation of T cell and NK cell proliferation.

The present invention further provides a variety of other polypeptidefusions [and related multimeric proteins comprising one or morepolypeptide fusions]. For example, a IL-21 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584 and derivatised according to the invention.Preferred dimerizing proteins in this regard include immunoglobulinconstant region domains. Immunoglobulin-IL-21 polypeptide fusions can beexpressed in genetically engineered cells. Auxiliary domains can befused to IL-21 polypeptides to target them to specific cells, tissues,or macromolecules (e.g., collagen).

The petides of the present invention, including full-length peptides,peptide fragments (e.g. ligand-binding fragments), and fusionpolypeptides can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed.(Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), and Ausubel et al., ibid.

It is to be recognized that according to the present invention, when acDNA is claimed as described above, it is understood that what isclaimed are both the sense strand, the anti-sense strand, and the DNA asdouble-stranded having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Also claimed is themessenger RNA (mRNA) which encodes the polypeptides of the presentinvention, and which mRNA is encoded by the above-described cDNA. Amessenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined above, with the exception that each thymine nucleotide (T)is replaced by a uracil nucleotide (U).

To direct an IL-21 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the protein, or may bederived from another secreted protein (e.g.,) or synthesized de novo.The secretory signal sequence is joined to the IL-21 DNA sequence in thecorrect reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the polypeptide of interest,although certain signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e. g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

IL-21 and variants thereof may be expressed in E-coli as described in WO04/55168. Optionally IL-21 variants may be produced by recombinant DNAtechniques in other organismes. To this end, DNA sequences encodinghuman IL-21 related polypeptides or IL-21 variants may be isolated bypreparing a genomic or cDNA library and screening for DNA sequencescoding for all or part of the protein by hybridization using syntheticoligonucleotide probes in accordance with standard techniques (cf.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989). For the presentpurpose, the DNA sequence encoding the protein is preferably of humanorigin, i.e. derived from a human genomic DNA or cDNA library.

The DNA sequences encoding the IL-21 variants may also be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by Beaucage and Caruthers, Tetrahedron Letters 22(1981), 1859-1869, or the method described by Matthes et al., EMBOJournal 3 (1984), 801-805. According to the phosphoamidite method,oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer,purified, annealed, ligated and cloned in suitable vectors.

The invention also comprises chemical modifications of the IL-21polypeptide, variant thereof or fusion proteins comprising IL-21 orvariants thereof. The chemical modification comprises covalentmodifications with an organic agent capable of reacting with a selectedside chain or a terminal residue.

Examples of such modifications are wherein a lipophilic substituent isattached to one or more amino acid residues at a position relative tothe amino acid sequence of SEQ ID NO:1 or 2 as described above. It is tobe understood that an amino acid residues at the position relative tothe amino acid sequence of SEQ ID NO:2 may be any amino acid residue andnot only the amino acid residue naturally present at that position. Inone embodiment the lipophilic substituent is attached to a lysine.

One or more of the lysines in IL-21 could be derivatives as described inthe application. In other preferred embodiments, additional lysines aresubstituted, inserted into the sequence or added at the N-terminal orC-terminal, and then optionally derivatised. Other aspects of theinvention includes addition of asp, glu, cys, gin, ser, thr, or tyrwhich carries function groups in the side chain for derivatising.

Preferred regions of insertions are where the overall activity of theprotein is not adversely affected. N-terminal and C-terminal truncationsmay occur simultaneously as well as additions in the terminal ofappropiate sequences.

In aspects of the invention any of the following positions are selectedfor substitution optionally in combination: Lys22, Lys53, Lys57, Lys76,Lys78, Lys89, Lys102, Lys103, Lys106, Lys113 or Lys118.

In an aspect of the invention the following substituents may bederivatised, optionally in combination and optionally after preparingvariants with Lysine in the corresponding positions; Arg66, Arg86,Arg87, Arg91, Arg111 or Arg127. All of the above positions arecalculated from the positions of the IL-21 peptide as described inWO00/53761 without the initial N-terminal sequence of 28 amino acids.

The term “lipophilic substituent” is characterised by comprising 4-40carbon atoms and having a solubility in water at 20° C. in the rangefrom about 0.1 mg/100 ml water to about 250 mg/100 ml water, such as inthe range from about 0.3 mg/100 ml water to about 75 mg/100 ml water.For instance, octanoic acid (C8) has a solubility in water at 20° C. of68 mg/100 ml, decanoic acid (C10) has a solubility in water at 20° C. of15 mg/100 ml, and octadecanoic acid (C18) has a solubility in water at20° C. of 0.3 mg/100 ml.

To obtain a satisfactory protracted profile of action of the IL-21derivative, the lipophilic substituent attached to the IL-21 moiety, asan example comprises 4-40 carbon atoms, such as 8-25 carbon atoms. Thelipophilic substituent may be attached to an amino group of the IL-21moiety by means of a carboxyl group of the lipophilic substituent whichforms an amide bond with an amino group of the amino acid to which it isattached. As an alternative, the lipophilic substituent may be attachedto said amino acid in such a way that an amino group of the lipophilicsubstituent forms an amide bond with a carboxyl group of the amino acid.As a further option, the lipophililic substituent may be linked to theIL-21 moiety via an ester bond. Formally, the ester can be formed eitherby reaction between a carboxyl group of the IL-21 moiety and a hydroxylgroup of the substituent-to-be or by reaction between a hydroxyl groupof the IL-21 moiety and a carboxyl group of the substituent-to-be. As afurther alternative, the lipophilic substituent can be an alkyl groupwhich is introduced into a primary amino group of the IL-21 moiety

In one embodiment of the invention the IL-21 derivative only has onelipophilic substituent attached to the IL-21 peptide.

In one embodiment of the invention the lipophilic substituent comprisesfrom 4 to 40 carbon atoms.

In one embodiment of the invention the lipophilic substituent comprisesfrom 8 to 25 carbon atoms.

In one embodiment of the invention the lipophilic substituent comprisesfrom 12 to 20 carbon atoms.

In one embodiment of the invention the lipophilic substituent isattached to an amino acid residue in such a way that a carboxyl group ofthe lipophilic substituent forms an amide bond with an amino group ofthe amino acid residue.

In other preferred embodiments, additional lysines are substituted,inserted into the sequence or added at the N-terminal or C-terminal, andthen optionally derivatised.

Preferred regions of insertions are where the overall activity of theprotein is not adversely affected. Preferred regions are the positionslisted above.

In one embodiment of the invention the lipophilic substituent isattached to an amino acid residue in such a way that an amino group ofthe lipophilic substituent forms an amide bond with a carboxyl group ofthe amino acid residue.

In one embodiment of the invention the lipophilic substituent isattached to the IL-21 peptide by means of a spacer.

In one embodiment of the invention the spacer is an unbranched alkanea,co-dicarboxylic acid group having from 1 to 7 methylene groups, suchas two methylene groups which spacer forms a bridge between an aminogroup of the IL-21 peptide and an amino group of the lipophilicsubstituent.

In one embodiment of the invention the spacer is an amino acid residueexcept a Cys residue, or a dipeptide. Examples of suitable spacersincludes β-alanine, gamma-aminobutyric acid (GABA), γ-glutamic acid,succinic acid, Lys, Glu or Asp, or a dipeptide such as Gly-Lys. When thespacer is succinic acid, one carboxyl group thereof may form an amidebond with an amino group of the amino acid residue, and the othercarboxyl group thereof may form an amide bond with an amino group of thelipophilic substituent. When the spacer is Lys, Glu or Asp, the carboxylgroup thereof may form an amide bond with an amino group of the aminoacid residue, and the amino group thereof may form an amide bond with acarboxyl group of the lipophilic substituent. When Lys is used as thespacer, a further spacer may in some instances be inserted between theε-amino group of Lys and the lipophilic substituent. In one embodiment,such a further spacer is succinic acid which forms an amide bond withthe ε-amino group of Lys and with an amino group present in thelipophilic substituent. In another embodiment such a further spacer isGlu or Asp which forms an amide bond with the ε-amino group of Lys andanother amide bond with a carboxyl group present in the lipophilicsubstituent, that is, the lipophilic substituent is a N^(ε)-acylatedlysine residue.

In one embodiment of the invention the spacer is selected from the listconsisting of β-alanine, gamma-aminobutyric acid (GABA), γ-glutamicacid, Lys, Asp, Glu, a dipeptide containing Asp, a dipeptide containingGlu, or a dipeptide containing Lys. In one embodiment of the inventionthe spacer is β-alanine. In one embodiment of the invention the spaceris gamma-aminobutyric acid (GABA). In one embodiment of the inventionthe spacer is γ-glutamic acid.

In one embodiment of the invention a carboxyl group of the parent IL-21peptide forms an amide bond with an amino group of a spacer, and thecarboxyl group of the amino acid or dipeptide spacer forms an amide bondwith an amino group of the lipophilic substituent.

In one embodiment of the invention an amino group of the parent IL-21peptide forms an amide bond with a carboxylic group of a spacer, and anamino group of the spacer forms an amide bond with a carboxyl group ofthe lipophilic substituent.

In one embodiment of the invention the lipophilic substituent comprisesa partially or completely hydrogenated cyclopentanophenathrene skeleton.

In one embodiment of the invention the lipophilic substituent is anstraight-chain or branched alkyl group. In one embodiment of theinvention the lipophilic substituent is the acyl group of astraight-chain or branched fatty acid.

In one embodiment of the invention the acyl group of a lipophilicsubstituent is selected from the group comprising CH₃(CH₂)_(n)CO—,wherein n is 4 to 38, such as CH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—,CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—,CH₃(CH₂)₂₀CO— and CH₃(CH₂)₂₂CO—.

In one embodiment of the invention the lipophilic substituent is an acylgroup of a straight-chain or branched alkane α,ω-dicarboxylic acid.

In one embodiment of the invention the acyl group of the lipophilicsubstituent is selected from the group comprising HOOC(CH₂)_(m)CO—,wherein m is 4 to 38, such as HOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—,HOOC(CH₂)₁₈CO—, HOOC(CH₂)₂₀CO— and HOOC(CH₂)₂₂CO—.

In one embodiment of the invention the lipophilic substituent is a groupof the formula CH₃(CH₂)_(p)((CH₂)_(q)COOH)CHNH—CO(CH₂)₂CO—, wherein pand q are integers and p+q is an integer of from 8 to 40, such as from12 to 35.

In one embodiment of the invention the lipophlic substituent is a groupof the formula CH₃(CH₂)_(r)CO—NHCH(COOH)(CH₂)₂CO—, wherein r is aninteger of from 10 to 24.

In one embodiment of the invention the lipophilic substituent is a groupof the formula CH₃(CH₂)_(s)CO—NHCH((CH₂)₂COOH)CO—, wherein s is aninteger of from 8 to 24.

In one embodiment of the invention the lipophilic substituent is a groupof the formula COOH(CH₂)_(t)CO— wherein t is an integer of from 8 to 24.

In one embodiment of the invention the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)_(u)CH₃, wherein u is aninteger of from 8 to 18.

In one embodiment of the invention the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—COCH((CH₂)₂COOH)NH—CO(CH₂)_(w)CH₃,wherein w is an integer of from 10 to 16.

In one embodiment of the invention the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NH—CO(CH₂)_(x)CH₃,wherein x is an integer of from 10to 16.

In one embodiment of the invention the lipophilic substituent is a groupof the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NHCO(CH₂)_(y)CH₃,wherein y is zero or an integer of from 1 to 22.

In one embodiment of the invention the lipophilic substituent isN-Lithocholoyl.

In one embodiment of the invention the lipophilic substituent isN-Choloyl.

In one embodiment of the invention the IL-21 derivative has onelipophilic substituent. In one embodiment of the invention the IL-21derivative has two lipophilic substituents. In one embodiment of theinvention the IL-21 derivative has three lipophilic substituents. In oneembodiment of the invention the IL-21 derivative has four lipophilicsubstituents.

The methods of the present invention also contemplate using chemicallymodified IL-21 compositions, in which an IL-21 polypeptide is linkedwith a polymeric molecule. Illustrative IL-21 polypeptides are solublepolypeptides that lack a functional transmembrane domain, such as amature IL-21 polypeptide. Typically, the polymer is water soluble sothat the IL-21 conjugate does not precipitate in an aqueous environment,such as a physiological environment. An example of a suitable polymer isone that has been modified to have a single reactive group, such as anactive ester for acylation, or an aldehyde for alkylation, In this way,the degree of polymerization can be controlled. An example of a reactivealdehyde is polyethylene glycol propionaldehyde, or mono-(C1-C10)alkoxy, or aryloxy derivatives thereof (see, for example, Harris, etal., U.S. Pat. No. 5,252,714). The polymer may be branched orunbranched. Moreover, a mixture of polymers can be used to produce IL-21conjugates. IL-21 conjugates used for therapy can comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Different sizes of PEG are described above.

In an aspect of the invention the peptide is derivatised with aN-terminal PEG group by oxidation of a serine with sodium periodate,followed by reaction of PEG-derivative, to which a hydroxylamine wasattached, yielding an oxime. In principle, the serine could also havebeen an internal serine residue or an added serine residue as describedabove.

Other methods for attaching PEG groups are described in G. Pasut, A.Guiotto, F. M. Veronese Expert Opin. Ther. Patents 2004, 14, 859-894.Variants of IL-21 suitable for attachment of polymeric groups may beobtained as described above.

In an aspect of the invention IL-21 is attached in the C-terminal of thepeptide. This may be achieved by using IL-21 or a variant of IL-21suitable as a substrate for CPY (carboxypeptidase Y) of which part ofthe reaction is described in in EP243929. This intermediate may then befurther substituted by a compound containing one or more reactivegroups, X, suitable for further substitution of with moleculescontaining the reactive group Y. The reactive group may be selected fromthe groups mentioned below.

In one embodiment the functional groups of X and Y are selected fromamongst carbonyl groups, such as keto and aldehyde groups, and aminoderivatives, such as hydrazine derivatives —NH—NH₂, hydrazinecarboxylate —O—C(O)—NH—NH₂, derivatives semicarbazide derivatives—NH—C(O)—NH—NH₂, thiosemicarbazide —NH—C(S)—NH—NH₂, derivatives carbonicacid dihydrazide —NHC(O)—NH—NH—C(O)—NH—NH₂, derivatives carbazidederivatives —NH—NH—C(O)—NH—NH₂, thiocarbazide derivatives—NH—NH—C(S)—NH—NH₂, aryl hydrazine derivatives —NH—C(O)—C₆H₄—NH—NH₂, andhydrazide derivatives —C(O)—NH—NH₂;oxylamine derivatives, such as —O—NH₂, —C(O)—O—NH₂, —NH—C(O)—O—NH₂ and—NH—C(S)—O—NH₂.

It is to be understood, that if the functional group comprised in X is acarbonyl group, then the functional group comprised in Y is an aminederivative, and vice versa. Due to the presence of —NH₂ groups in mostpeptides, a better selectivity is believed to be obtained if X comprisesa keto- or an aldehyde-functionality.

Examples of derivatives of PEG suitable in the reaction described aboveare

wherein n is 1,2, 3, 4, 5 or 6 and mPEG has a molecular weight of 10kDa, 20 kDa, 30 kDa or 40 kDa.

wherein m is 1, 2, 3, 4, 5 or 6 and mPEG has a molecular weight of 10kDa, 20 kDa, 30 kDa or 40 kDa.

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein n is 0,1,2,3,4,5 or 6 and m is 1, 2,3, 4, 5 or 6 and mPEG has amolecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein n is 1, 2, 3, 4, 5 or 6 and mPEG has a molecular weight of 10kDa, 20 kDa, 30 kDa or 40 kDa,

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,

wherein Y is —O—NH₂, NH—NH₂,

n, m and s are any number from 0 to 20;

R′ and R″ independently represents for example methyl, phenyl, biphenyl,phenoxyphenyl, phenylcarboxyphenyl.

At any suitable position in the alkyl chains in any of the formulasabove a group of the formula —SO₂—, —C(O)NH—, —C(O)NHSO₂—, —SO₂-phenyl-,C(O)NHSO₂-phenyl- may be inserted in either direction. Optionally thegroup C(O)NH in the above formula may be substituted by

In an embodiment of the invention the introduction of the derivative isintroduced in one step. The R—X then contains the derivatives to beintroduced into IL-21. The nucleophile represents for example aminoacids, which has been modified to carry the derivative. In principle anysequence of amino acids may be used. In an aspect of the inventionnucleophiles such as G(₁₋₅)-PEG, G(₁₋₅)-lipid. G(₁₋₄) —NH—CH₂—CHO,G(₁₋₄) —NH—CH₂—C—O—NH₂ etc. are used.

PEG is a suitable polymer molecule, since it has only few reactivegroups capable of cross-linking compared to polysaccharides such asdextran. In particular, monofunctional PEG, e.g. methoxypolyethyleneglycol (mPEG), is of interest since its coupling chemistry is relativelysimple (only one reactive group is available for conjugating withattachment groups on the polypeptide). Consequently, the risk ofcross-linking is eliminated, the resulting polypeptide conjugates aremore homogeneous and the reaction of the polymer molecules with thepolypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule areprovided in activated form, i.e. with reactive functional groups.Suitable activated polymer molecules are commercially available, e.g.from Shearwater Corp., Huntsville, Ala., USA, or from PolyMASCPharmaceuticals pic, UK. Alternatively, the polymer molecules can beactivated by conventional methods known in the art, e.g. as disclosed inWO 90/13540. Specific examples of activated linear or branched polymermolecules for use in the present invention are described in theShearwater Corp. 1997 and 2000 Catalogs (Functionalized BiocompatiblePolymers for Research and pharmaceuticals, Polyethylene Glycol andDerivatives, incorporated herein by reference). Specific examples ofactivated PEG polymers include the following linear PEGs: NHS-PEG (e.g.SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, andSCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG,ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs suchas PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat.No. 5,643,575, both of which are incorporated herein by reference.Furthermore, the following publications, incorporated herein byreference, disclose useful polymer molecules and/or PEGylationchemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No.5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673,EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503and EP 154 316.

The conjugation of the polypeptide and the activated polymer moleculesis conducted by use of any conventional method, e.g. as described in thefollowing references (which also describe suitable methods foractivation of polymer molecules): R. F. Taylor, (1991), “Proteinimmobilisation. Fundamental and applications”, Marcel Dekker, N.Y.; S.S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”,CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “ImmobilizedAffinity Ligand Techniques”, Academic Press, N.Y.). The skilled personwill be aware that the activation method and/or conjugation chemistry tobe used depends on the attachment group(s) of the polypeptide (examplesof which are given further above), as well as the functional groups ofthe polymer (e.g. being amine, hydroxyl, carboxyl, aldehyde, sulfydryl,succinimidyl, maleimide, vinysulfone or haloacetate). The PEG-ylationmay be directed towards conjugation to all available attachment groupson the polypeptide (i.e. such attachment groups that are exposed at thesurface of the polypeptide) or may be directed towards one or morespecific attachment groups, e.g. the N-terminal amino group (U.S. Pat.No. 5,985,265). Furthermore, the conjugation may be achieved in one stepor in a stepwise manner (e.g. as described in WO 99/55377).

It will be understood that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form of such molecules (e.g. whether they arelinear or branched), and where in the polypeptide such molecules areattached. The molecular weight of the polymer to be used will be chosentaking into consideration the desired effect to be achieved. Forinstance, if the primary purpose of the conjugation is to achieve aconjugate having a high molecular weight and larger size (e.g. to reducerenal clearance), one may choose to conjugate either one or a few highmolecular weight polymer molecules or a number of polymer molecules witha smaller molecular weight to obtain the desired effect. Preferably,however, several polymer molecules with a lower molecular weight will beused. This is also the case if a high degree of epitope shielding isdesired. In such cases, 2-8 polymers with a molecular weight of e.g.about 5,000 Da, such as 3-6 such polymers, may for example be used. Asthe examples below illustrate, it may be advantageous to have a largernumber of polymer molecules with a lower molecular weight (e.g. 4-6 witha MW of 5000) compared to a smaller number of polymer molecules with ahigher molecular weight (e.g. 1-3 with a MW of 12,000-20,000) in termsof improving the functional in vivo half-life of the polypeptideconjugate, even where the total molecular weight of the attached polymermolecules in the two cases is the same or similar. It is believed thatthe presence of a larger number of smaller polymer molecules providesthe polypeptide with a larger diameter or apparent size than e.g. asingle yet larger polymer molecule, at least when the polymer moleculesare relatively uniformly distributed on the polypeptide surface. It hasfurther been found that advantageous results are obtained when theapparent size (also referred to as the “apparent molecular weight” or“apparent mass”) of at least a major portion of the conjugate of theinvention is at least about 50 kDa, such as at least about 55 kDa, suchas at least about 60 kDa, e.g. at least about 66 kDa. This is believedto be due to the fact that renal clearance is substantially eliminatedfor conjugates having a sufficiently large apparent size. In the presentcontext, the “apparent size” of a IL-21 conjugate or IL-21 polypeptideis determined by the SDS-PAGE method.

In an embodiment of the invention PEG is conjugated to a peptideaccording to the present invention may be of any molecular weight. Inparticular the molecular weight may be between 500 and 100,000 Da, suchas between 500 and 60,000 Da, such as between 1000 and 40,000 Da, suchas between 5,000 and 40,000 Da. In particular, PEG with molecularweights of 10,000 Da, 20,000 Da or 40,000 KDa may be used in the presentinvention. In all cases the PEGs may be linear or branched. In anembodiment of the invention the PEG groups are 5 kDa, 10 kDa, 20 kDa, 30kDa, 40 kDa og 60 kDa.

In an embodiment of the invention, one or more polymeric molecules areadded to the peptide.

The present invention provides compounds which are suitable forattachment of a polymeric group. In an embodiment of the invention thederivative thus provides a peptide which has an improved in vivo halflife. This may be achieved by protecting the compound against chemicaldegradation, proteolytic degradation, or antibody recognition—or anyother mechanisms.

In an embodiment the compounds provided are less toxic.

In an embodiment the compounds provided are more water soluble

In an embodiment the compounds provided has a modified biodistribution.

The above are with reference to the non-derivatised analogues or to thehIL-21.

Pharmaceutical Compositions

The invention provides in a particular embodiment the following:

Another object of the present invention is to provide a pharmaceuticalformulation comprising IL-21, analogues or derivatives thereof, oroptionally together with any other compound mentioned in the presentapplication which is present in a concentration from 0.1 mg/ml to 100mg/ml, and wherein said formulation has a pH from 2.0 to 10.0. Theformulation may further comprise a buffer system, preservative(s),tonicity agent(s), chelating agent(s), stabilizers and surfactants. Inone embodiment of the invention the pharmaceutical formulation is anaqueous formulation, i.e. formulation comprising water. Such formulationis typically a solution or a suspension. In a further embodiment of theinvention the pharmaceutical formulation is an aqueous solution. Theterm “aqueous formulation” is defined as a formulation comprising atleast 50% w/w water. Likewise, the term “aqueous solution” is defined asa solution comprising at least 50% w/w water, and the term “aqueoussuspension” is defined as a suspension comprising at least 50% w/wwater.

In another embodiment the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect the invention relates to a pharmaceuticalformulation comprising an aqueous solution of IL-21 or any othercompound as mentioned above and a buffer, wherein said compound ispresent in a concentration from 0.1 mg/ml or as mentioned above,preferably from 0.5 mg/ml-50 mg/ml and wherein said formulation has a pHfrom about 2.0 to about 10.0. Preferred pH is from 3.0 to about 8.0.Particular preferred range is from 4.0-6.0, such as for example theranges 4.0-4.5, 4.5-5.0, 5.0-5.5 and 5.5-6.0.

In another embodiment of the invention the pH of the formulation isselected from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, and 10.0.

In a further embodiment of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment of the invention the formulation furthercomprises a pharmaceutically acceptable antimicrobial preservative. In afurther embodiment of the invention the preservative is selected fromthe group consisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomersal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixturesthereof. In a further embodiment of the invention the preservative ispresent in a concentration from 0.1 mg/ml to 20 mg/ml. In a furtherembodiment of the invention the preservative is present in aconcentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of theinvention the preservative is present in a concentration from 5 mg/ml to10 mg/ml. In a further embodiment of the invention the preservative ispresent in a concentration from 10 mg/ml to 20 mg/ml. Each one of thesespecific preservatives constitutes an alternative embodiment of theinvention. The use of a preservative in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises an isotonic agent. In a further embodiment of the inventionthe isotonic agent is selected from the group consisting of a salt (e.g.sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In one embodiment the sugar additive is sucrose. Sugar alcohol isdefined as a C4-C8 hydrocarbon having at least one —OH group andincludes, for example, mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. In one embodiment the sugar alcoholadditive is mannitol. The sugars or sugar alcohols mentioned above maybe used individually or in combination. There is no fixed limit to theamount used, as long as the sugar or sugar alcohol is soluble in theliquid preparation and does not adversely effect the stabilizing effectsachieved using the methods of the invention. In one embodiment, thesugar or sugar alcohol concentration is between about 1 mg/ml and about150 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 1 mg/ml to 50 mg/ml. In a furtherembodiment of the invention the isotonic agent is present in aconcentration from 1 mg/ml to 7 mg/ml. In a further embodiment of theinvention the isotonic agent is present in a concentration from 8 mg/mlto 24 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 25 mg/ml to 50 mg/ml. Each one ofthese specific isotonic agents constitutes an alternative embodiment ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises a chelating agent. In a further embodiment of the inventionthe chelating agent is selected from salts of ethylenediaminetetraaceticacid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In afurther embodiment of the invention the chelating agent is present in aconcentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of theinvention the chelating agent is present in a concentration from 0.1mg/ml to 2 mg/ml. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/ml to 5 mg/ml. Each one ofthese specific chelating agents constitutes an alternative embodiment ofthe invention. The use of a chelating agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises a stabilizer. The use of a stabilizer in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a polypeptide that possibly exhibits aggregate formation duringstorage in liquid pharmaceutical formulations. By “aggregate formation”is intended a physical interaction between the polypeptide moleculesthat results in formation of oligomers, which may remain soluble, orlarge visible aggregates that precipitate from the solution. By “duringstorage” is intended a liquid pharmaceutical composition or formulationonce prepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or formulation is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli (1984) J.Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) inSpray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez,U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm.18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), orair drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser(1991) Biopharm. 4:47-53). Aggregate formation by a polypeptide duringstorage of a liquid pharmaceutical composition can adversely affectbiological activity of that polypeptide, resulting in loss oftherapeutic efficacy of the pharmaceutical composition. Furthermore,aggregate formation may cause other problems such as blockage of tubing,membranes, or pumps when the polypeptide-containing pharmaceuticalcomposition is administered using an infusion system.

The pharmaceutical compositions of the invention may further comprise anamount of an amino acid base sufficient to decrease aggregate formationby the polypeptide during storage of the composition. By “amino acidbase” is intended an amino acid or a combination of amino acids, whereany given amino acid is present either in its free base form or in itssalt form. Where a combination of amino acids is used, all of the aminoacids may be present in their free base forms, all may be present intheir salt forms, or some may be present in their free base forms whileothers are present in their salt forms. In one embodiment, amino acidsto use in preparing the compositions of the invention are those carryinga charged side chain, such as arginine, lysine, aspartic acid, andglutamic acid. Any stereoisomer (i.e., L, D, or DL isomer) of aparticular amino acid (e.g. glycine, methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine andmixtures thereof) or combinations of these stereoisomers, may be presentin the pharmaceutical compositions of the invention so long as theparticular amino acid is present either in its free base form or itssalt form. In one embodiment the L-stereoisomer is used. Compositions ofthe invention may also be formulated with analogues of these aminoacids. By “amino acid analogue” is intended a derivative of thenaturally occurring amino acid that brings about the desired effect ofdecreasing aggregate formation by the polypeptide during storage of theliquid pharmaceutical compositions of the invention. Suitable arginineanalogues include, for example, aminoguanidine, ornithine andN-monoethyl L-arginine, suitable methionine analogues include ethionineand buthionine and suitable cysteine analogues include S-methyl-Lcysteine. As with the other amino acids, the amino acid analogues areincorporated into the compositions in either their free base form ortheir salt form. In a further embodiment of the invention the aminoacids or amino acid analogues are used in a concentration, which issufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention methionine (or other sulphuricamino acids or amino acid analogous) may be added to inhibit oxidationof methionine residues to methionine sulfoxide when the polypeptideacting as the therapeutic agent is a polypeptide comprising at least onemethionine residue susceptible to such oxidation. By “inhibit” isintended minimal accumulation of methionine oxidized species over time.Inhibiting methionine oxidation results in greater retention of thepolypeptide in its proper molecular form. Any stereoisomer of methionine(L, D, or DL isomer) or combinations thereof can be used. The amount tobe added should be an amount sufficient to inhibit oxidation of themethionine residues such that the amount of methionine sulfoxide isacceptable to regulatory agencies. Typically, this means that thecomposition contains no more than about 10% to about 30% methioninesulfoxide. Generally, this can be achieved by adding methionine suchthat the ratio of methionine added to methionine residues ranges fromabout 1:1 to about 1000:1, such as 10:1 to about 100:1.

In a further embodiment of the invention the formulation furthercomprises a stabilizer selected from the group of high molecular weightpolymers or low molecular compounds. In a further embodiment of theinvention the stabilizer is selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). Each one of these specificstabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizingagents, which further enhance stability of a therapeutically activepolypeptide therein. Stabilizing agents of particular interest to thepresent invention include, but are not limited to, methionine and EDTA,which protect the polypeptide against methionine oxidation, and anonionic surfactant, which protects the polypeptide against aggregationassociated with freeze-thawing or mechanical shearing.

In a further embodiment of the invention the formulation furthercomprises a surfactant. In a further embodiment of the invention thesurfactant is selected from a detergent, ethoxylated castor oil,polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fattyacid esters, polyoxypropylene-polyoxyethylene block polymers (eg.poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100 ),polyoxyethylene sorbitan fatty acid esters, polyoxyethylene andpolyethylene derivatives such as alkylated and alkoxylated derivatives(tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglyceridesor ethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, alcohols, glycerol, lectins and phospholipids (eg.phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin),derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) andlysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, andglycerophospholipids (eg. cephalins), glyceroglycolipids (eg.galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acidderivatives-(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulphate or sodium lauryl sulphate),sodium caprylate, cholic acid or derivatives thereof, bile acids andsalts thereof and glycine or taurine conjugates, ursodeoxycholic acid,sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate,anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyidimethylammonio-1-propanesulfonate, cationicsurfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), non-ionic surfactants (eg.Dodecyl-D-glucopyranoside), poloxamines (eg. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof. Each one of these specific surfactants constitutes analternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing IL-21 or any other compound asmentioned above according to the present invention may be administeredto a patient in need of such treatment at several sites, for example, attopical sites, for example, skin and mucosal sites, at sites whichbypass absorption, for example, administration in an artery, in a vein,in the heart, and at sites which involve absorption, for example,administration in the skin, under the skin, in a muscle or in theabdomen.

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, rinses, capsules, for example, hardgelatine capsules and soft gelatine capsules, suppositories, rectalcapsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops,ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginalrings, vaginal ointments, injection solution, in situ transformingsolutions, for example in situ gelling, in situ setting, in situprecipitating, in situ crystallization, infusion solution, and implants.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of of IL-21 or anyother compound as mentioned above, increase bioavailability, increasesolubility, decrease adverse effects, achieve chronotherapy well knownto those skilled in the art, and increase patient compliance or anycombination thereof. Examples of carriers, drug delivery systems andadvanced drug delivery systems include, but are not limited to,polymers, for example cellulose and derivatives, polysaccharides, forexample dextran and derivatives, starch and derivatives, poly(vinylalcohol), acrylate and methacrylate polymers, polylactic andpolyglycolic acid and block co-polymers thereof, polyethylene glycols,carrier proteins, for example albumin, gels, for example, thermogellingsystems, for example block co-polymeric systems well known to thoseskilled in the art, micelles, liposomes, microspheres, nanoparticulates,liquid crystals and dispersions thereof, L2 phase and dispersions thereof, well known to those skilled in the art of phase behaviour inlipid-water systems, polymeric micelles, multiple emulsions,self-emulsifying, self-microemulsifying, cyclodextrins and derivativesthereof, and dendrimers.

Compositions of the current invention are useful in the formulation ofsolids, semisolids, powder and solutions for pulmonary administration ofIL-21 or any other compound as mentioned above using, for example ametered dose inhaler, dry powder inhaler and a nebulizer, all beingdevices well known to those skilled in the art.

Compositions of the current invention are specifically useful in theformulation of controlled, sustained, protracting, retarded, and slowrelease drug delivery systems. More specifically, but not limited to,compositions are useful in formulation of parenteral controlled releaseand sustained release systems (both systems leading to a many-foldreduction in number of administrations), well known to those skilled inthe art. Even more preferably, are controlled release and sustainedrelease systems administered subcutaneous. Without limiting the scope ofthe invention, examples of useful controlled release system andcompositions are hydrogels, oleaginous gels, liquid crystals, polymericmicelles, microspheres, nanoparticles,

Methods to produce controlled release systems useful for compositions ofthe current invention include, but are not limited to, crystallization,condensation, co-crystallization, precipitation, co-precipitation,emulsification, dispersion, high pressure homogenisation, encapsulation,spray drying, microencapsulating, coacervation, phase separation,solvent evaporation to produce microspheres, extrusion and supercriticalfluid processes. General reference is made to Handbook of PharmaceuticalControlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) andDrug and the Pharmaceutical Sciences vol. 99: Protein Formulation andDelivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a solution or suspension for theadministration of IL-21 or any other compound as mentioned above, in theform of a nasal or pulmonal spray. As a still further option, thepharmaceutical compositions containing IL-21 or any other compound asmentioned above can also be adapted to transdermal administration, e.g.by needle-free injection or from a patch, optionally an iontophoreticpatch, or transmucosal, e.g. buccal, administration.

The term “stabilized formulation” refers to a formulation with increasedphysical stability, increased chemical stability or increased physicaland chemical stability.

The term “physical stability” of the protein formulation as used hereinrefers to the tendency of the protein to form biologically inactiveand/or insoluble aggregates of the protein as a result of exposure ofthe protein to thermo-mechanical stresses and/or interaction withinterfaces and surfaces that are destabilizing, such as hydrophobicsurfaces and interfaces. Physical stability of the aqueous proteinformulations is evaluated by means of visual inspection and/or turbiditymeasurements after exposing the formulation filled in suitablecontainers (e.g. cartridges or vials) to mechanical/physical stress(e.g. agitation) at different temperatures for various time periods.Visual inspection of the formulations is performed in a sharp focusedlight with a dark background. The turbidity of the formulation ischaracterized by a visual score ranking the degree of turbidity forinstance on a scale from 0 to 3 (a formulation showing no turbiditycorresponds to a visual score 0, and a formulation showing visualturbidity in daylight corresponds to visual score 3). A formulation isclassified physically unstable with respect to protein aggregation, whenit shows visual turbidity in daylight. Alternatively, the turbidity ofthe formulation can be evaluated by simple turbidity measurementswell-known to the skilled person. Physical stability of the aqueousprotein formulations can also be evaluated by using a spectroscopicagent or probe of the conformational status of the protein. The probe ispreferably a small molecule that preferentially binds to a non-nativeconformer of the protein. One example of a small molecular spectroscopicprobe of protein structure is Thioflavin T. Thioflavin T is afluorescent dye that has been widely used for the detection of amyloidfibrils. In the presence of fibrils, and perhaps other proteinconfigurations as well, Thioflavin T gives rise to a new excitationmaximum at about 450 nm and enhanced emission at about 482 nm when boundto a fibril protein form. Unbound Thioflavin T is essentiallynon-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in proteinstructure from native to non-native states. For instance the“hydrophobic patch” probes that bind preferentially to exposedhydrophobic patches of a protein. The hydrophobic patches are generallyburied within the tertiary structure of a protein in its native state,but become exposed as a protein begins to unfold or denature. Examplesof these small molecular, spectroscopic probes are aromatic, hydrophobicdyes, such as antrhacene, acridine, phenanthroline or the like. Otherspectroscopic probes are metal-amino acid complexes, such as cobaltmetal complexes of hydrophobic amino acids, such as phenylalanine,leucine, isoleucine, methionine, and valine, or the like.

The term “chemical stability” of the protein formulation as used hereinrefers to chemical covalent changes in the protein structure leading toformation of chemical degradation products with potential lessbiological potency and/or potential increased immunogenic propertiescompared to the native protein structure. Various chemical degradationproducts can be formed depending on the type and nature of the nativeprotein and the environment to which the protein is exposed. Eliminationof chemical degradation can most probably not be completely avoided andincreasing amounts of chemical degradation products is often seen duringstorage and use of the protein formulation as well-known by the personskilled in the art. Most proteins are prone to deamidation, a process inwhich the side chain amide group in glutaminyl or asparaginyl residuesis hydrolysed to form a free carboxylic acid. Other degradation pathwaysinvolves formation of high molecular weight transformation productswhere two or more protein molecules are covalently bound to each otherthrough transamidation and/or disulfide interactions leading toformation of covalently bound dimer, oligomer and polymer degradationproducts (Stability of Protein Pharmaceuticals, Ahern. T. J. & ManningM. C., Plenum Press, New York 1992). Oxidation (of for instancemethionine residues) can be mentioned as another variant of chemicaldegradation. The chemical stability of the protein formulation can beevaluated by measuring the amount of the chemical degradation productsat various time-points after exposure to different environmentalconditions (the formation of degradation products can often beaccelerated by for instance increasing temperature). The amount of eachindividual degradation product is often determined by separation of thedegradation products depending on molecule size and/or charge usingvarious chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a “stabilized formulation” refers to aformulation with increased physical stability, increased chemicalstability or increased physical and chemical stability. In general, aformulation must be stable during use and storage (in compliance withrecommended use and storage conditions) until the expiration date isreached.

EXAMPLES

Recombinant Interleukin 21 (IL21) was expressed as inclusion bodies inE.coli as described in WO04/55168 with a N-terminal extension(Met-Ser-hIL21). The N-terminal Met residue is removed by the proteasesystems present in E.coli, leaving Ser-hIL21. The Glu-Ala-Glu amino acidsequence can be present or absent.

The protein was refolded and purified to 90-95% purity usingconventional chromatographic methods.

The pure protein was subsequently N-terminally PEGylated via oxidationof the N-terminal serine by reaction with sodium periodate, followed byreaction of PEG-derivative, to which a hydroxylamine was attached,yielding an oxime.

Subsequent purification was done using gelfiltration or size exclusionchromatography (SEC).

In an proliferation assay as for example the BAF3 assay described below,the pegylated IL21 was equipotent with the unpegylated standard,indicating that the pegylation does not interfere with receptor binding,and that the reaction procedure are not harmful to the protein.

PREPARATORY EXAMPLES

A method for the attachment of a PEG-moiety to the C-terminus of a IL-21derivative may be performed analogously to the attachment of chemicalmoieties to other peptides or proteins described above:

A PEG-moiety may be attached to the C-terminus of IL-21 or an IL-21derivative such as e.g. hIL-21, by a two step method.

In the first step, a suitable IL-21-analogue such as e.g.(hIL-21yl)alanine is subjected to a transpeptidation reaction catalyzedby carboxypeptidase Y (CPY) in the presence of a suitable nucleophilee.g. (S)-2-amino-3-(4-(propargyloxy)phenyl)propanoic acid in a suitablebuffer such as a HEPES/TMEDA-buffer at a suitable pH such e.g. pH 7.5 orpH8 at a suitable temperature such as e.g. room temperature 30° C. or35° C.

In the second step, a suitable derivatized PEG-reagent may be reactedwith (S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide.E.g. an excess of 4-(mPEG20000yl)-N-(3-(hydroxyimino)benzyl) butanoicamide may be reacted under oxidative conditions such as e.g. sodiumhypochlorite solution to 4-(mPEG20000yl)-N-3-(oxycyano)benzylbutanoicamide. A solution of 4-(mPEG20000yl)-N-(3-(oxycyano)benzylbutanoic amidemay be added to a solution of(S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide toyield(S)-2-((hIL-21yl)amino)-3-(4-((3-(3-((4-(mPEG20000yl)butanoylamino)methyl)phenyl)isoxazol-5-yl)methoxy)phenyl)propanoicamide.

4-(mPEG20000yl)-N-3-(hydroxyimino)benzyl) butanoic amide may be preparedfrom commercially available 3-((tert-butoxycarbonylamino)methyl)benzoicacid, which may be reduced with a suitable reagent or combination ofreagents e.g. in a two step procedure known to a person trained in theart, comprising in a first step addition of ethyl chloroformate in thepresence of a base such as e.g. triethylamine, removal of the formedtriethylammonium chloride by filtration and addition of lithiumborohydride to yield tert-butyl N-(3-(hydroxylmethyl)benzyl)carbamate.tert-Butyl N-(3-(hydroxylmethyl)benzyl)carbamate may be oxidized with asuitable reagent or combination of reagents, e.g. using a Swernoxidation, known to a person trained in the art, comprising the additionof a solution of the alcohol in e.g. dichloromethane at −78° C. to amixture of oxalyl chloride and dimethylsulfoxidein dichloromethane,which has been formed at −78° C., followed by addition of a suitableamino-base such as e.g. triethylamine and subsequent warming to roomtemperature. The formed tert-butyl N-(3-formylbenzyl)carbamate may bereacted to yield tert-butyl N-(3-((hydroxylimino)methyl)benzyl) byreaction with the free base or a suitable salt of hydroxylamine in asolution of e.g. sodium hydroxide in water. The BOC-protection group maybe removed from tert-butyl N-(3-((hydroxylimino)methyl)benzyl) bymethods described in the literature (e.g. T. W. Green, P. G. M WutsProtective groups in organic synthesis 2^(nd) ed. Wiley, New York, 1991)e.g. by treatment with a 50% solution of trifluoroacetic acid indichloromethane to give 3-(aminomethyl)benzaldehyde oxime. Finally,4-(mPEG20000yl)-N-(3-(hydroxyimino)benzyl)butanoic amide may be preparedby amide-forming reaction, comprising a reaction of the free base or asuitable salt of 3-(aminomethyl)benzaldehyde oxime in the presence of anexcess of a suitable base such as e.g. ethyidiisopropylamine withcommercially available 2,5-dioxypyrrolidinyl 4-(mPEG20000yl)butanoicester (Nektar, 2M450P01).

In an alternative second step, a mixture of an appropriate amount ofcopper sulphate penthydrate, e.g. 5% or 10% or 1 equivalent or 10equivalents with respect to(S)2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide and anappropriate amount of L-ascorbic acid, such as e.g. 50 eq with respectto (S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide, maybe prepared in water, which is buffered with 2,6-lutidine. After aappropriate period of time such as e.g 5 min, this solution may be givento a solution of(S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide andN-(2-(mPEG20000yl)ethyl) 11-azidoundecanoic amide which is buffered with2,6-lutidine. The reaction mixture may be kept at a appropriatetemperature such as e.g. room temperature until a mixture of a singlecompound selected from(S)-((hIL-21)amino)-3-(4-((1-(10-(N-(2-(mPEG20000yl)ethyl)carbamoyl)decanyl)-1,2,3-triazol-4-yl)methoxy)phenyl)and (S)-((hIL-21)amino)-3-(4-((1-(10-(N-(2-(mPEG20000yl)ethyl)carbamoyl)decanyl)-1,2,3-triazol-5-yl)methoxy)phenyl)and may be formed.

The synthesis of N-(2-(mPEG20000yl)ethyl)11-azidoundecanoic amide may beperformed by reaction of commercially available methyl11-bromoundecanoic ester with sodium azide in an appropriate solventsuch as e.g. N,N-dimethylformamide at an appropriate temperature as e.g.60° C. The formed methyl 11-azidoundecanoic ester may be saponified by amethod known to a person skilled in the art and described in theliterature (e.g. T. W. Green, P. G. M Wuts Protective groups in organicsynthesis 2^(nd) ed. Wiley, New York, 1991) such as e.g. potassiumhydroxide in methanol or potassium triethylsilanolate intetrahydrofuran. The resulting acid may be activated by a method knownto a person skilled in the art e.g. by reaction with2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) in anappropriate solvent such as e.g. N,N-dimethylformamide at an appropriatetemperature such as e.g. room temperature to give11-azido-N-2,5-dioxopyrrolidin-1-ylundecanoic amide.11-Azido-N-2,5-dioxopyrrolidin-1-ylundecanoic amide may be reacted withcommercially available (2-(mPEG20000yl)ethyl)amine (Nektar 2M2U0P01) inan appropriate solvent such as e.g. dichloromethane and in the presenceof an appropriate base such as e.g. triethylamine orethyldiisopropylamine go give N-(2-(mPEG20000yl)ethyl)11-azidoundecanoicamide.

EXAMPLE1-(((((4-((4-(mPEG-20000yl)butanoyl)amino)butoximinoacetyl)serinyl)glutamyl)alaninyl)glutamyl)hIL-21

Step 1:

2-(4-(tert-Butoxycarbonylaminoxy)butyl)isoindole-1,3-dione

To a mixture of commercially available N-(4-bromobutyl)phthalimide (2.82g, 10 mmol) and N-Boc-hydroxylamine (2.08 g, 15.6 mmol) was addedacetonitrile (2 ml) and successively 1,8-diazabicyclo[5.4.0]undec-7-ene(2.25 ml, 15 mmol). The reaction mixture was stirred at room temperaturefor 30 min and then at 50° C. for 2 days. It was diluted with a mixtureof water (30 ml) and 1 N hydrochloric acid (20 ml). It was extractedwith ethyl acetate (2×100 ml). The organic phase was washed with brine(50 ml) and was dried over magnesium sulphate. The crude product waspurified by chromatography on silica (60 g), using a gradient ofheptane/ethyl acetate 1:0 to 0:1 as eluent to give 2.08 g of2-(4-(tert-butoxycarbonylaminoxy)butyl)isoindole-1,3-dione.

Step 2:

N-(4-aminobutoxy)carbamic acid tert-butyl ester

Hydrazine hydrate (1.0 ml, 20 mmol) was added to a solution of2-(4-(tert-butoxycarbonylaminoxy)butyl)isoindole-1,3-dione (2.08 g, 6.22mmol) in ethanol (8.0 ml). The reaction mixture was stirred at 80° C.for 65 h. The solvent was removed in vacuo. The residue was dissolved intoluene (10 ml) and the solvent was removed in vacuo. The residue wassuspended in 1 N hydrochloric acid (10 ml). The precipitation wasremoved by filtration and was washed with water (2 ml). The filtrate andthe wash-liquids were combined and made basic with potassium carbonate.The solution was extracted with dichloromethane (4×20 ml). The organiclayer was dried over magnesium sulphate. The solvent was removed invacuo to give 0.39 g of N-(4-aminobutoxy)carbamic acid tert-butyl ester.Potassium carbonate (3 g) was added to the aqueous phase, which wasextracted with dichloromethane (3×20 ml). These combined organic layerswere dried over magnesium sulphate. The solvent was removed in vacuo togive another 0.39 g of N-(4-aminobutoxy)carbamic acid tert-butyl ester.

Step 3:

N-(4-(4-(mPEG20000yl)butanolyamino)butoxy)carbamic acid tert-butyl ester

The commercially available N-hydroxysuccinimide ester ofmPEG2000ylbutanoic acid (Nektar “mPEG-SBA”, #2M450P01, 3 g, 0.15 mmol)was dissolved in dichloromethane (25 ml). N-(4-Aminobutoxy)carbamic acidtert-butyl ester (0.12 g, 0.59 mmol) was added. The reaction mixture wasshaken at room temperature. Diethyl ether was added until aprecipitation was obtained. The precipitation was isolated byfiltration. The material was dried in vacuo to yield 2.39 g ofN-(4-(4-(mPEG20000yl)butanolyamino)butoxy)carbamic acid tert-butylester.

Step 4:

N-(4-Aminoxybutyl)-4-(mPEG20000yl)butanolyamide

Trifluoroacetic acid (20 ml) was added to a solution ofN-(4-(4-(mPEG20000yl)butanolyamino)butoxy)carbamic acid tert-butyl ester(2.39 g, 0.12 mmol) in dichloromethane (20 ml). The reaction mixture wasshaken for 30 min. Diethyl ether (100 ml) was added. The formedprecipitation was isolated by filtration. It was washed with diethylether (2×100 ml) and dried in vacuo to give 1.96 g ofN-(4-aminoxybutyl)-4-(mPEG20000yl)butanolyamide

Step 5:

1-((((Serinyl)glutamyl)alninyl)glutamyl)hIL-21 (4 mg, lyophilized in aphosphate-buffer, 252 nmol) was dissolved in 0.400 ml of a buffer,consisting of triethanolamine (0.008 ml) in water (4 ml). A solution ofmethionine (3.15 mg, 21420 nmol) in water (0.12 ml) and a solution ofsodium periodate (0.38 mg, 1890 nmol) were added successively. Thereaction mixture was left for 30 min at room temperature. A solution ofN-(4-aminoxybutyl)-4-(mPEG20000yl)butanolyamide (77 mg, 3780 nmol) inwater (0.240 ml) was added. The pH was adjusted to pH 4-5 with glacialacetic acid (0.004 ml). The reaction mixture was left at roomtemperature for 16 h. The reaction mixture was diluted with a solutionof triethanolamine (12 mg) in water (3.2 ml) and was kept at −18° C.until purification.

Protein Chemistry

Recombinant Interleukin 21 (IL21) was expressed as inclusion bodies inE.coli with a N-terminal extension (Met-Ser-Glu-Ala-Glu-hIL21). TheN-terminal Met residue is removed by the protease systems present inE.coli, leaving Ser-Glu-Ala-Glu-hIL21. The Glu-Ala-Glu amino acidsequence can be present or absent (we initiate experiments withMet-Ser-hIL21).

The protein was refolded and purified to 90-95% purity usingconventional chromatographic methods.

The pure protein was subsequently N-terminally pegylated via reactiondescribed in steps 1-5.

Subsequent purification was done using gelfiltration or size exclusionchromatography (SEC).

In an proliferation assay, the pegylated IL21 showed similar potentencyto the unpegylated standard, indicating that the pegylation does notinterfere with receptor binding, and that the reaction procedures arenot harmful to the protein.

Pharmacological Methods

Proliferation assay using Baf-3(IL21R) cells.

IL3 dependent Baf-3 cells transfected with either the murine or thehumane IL21 R are grown in IL-3 containing culture medium until setup ofa proliferation assay (preferably 3 days).

Cells used for the assay are washed in IL3-free medium and plated in 96well-plates with 50,000 c/w in assay media (without IL-3). IL21 is addedin serial dilutions from 10⁻⁷M-10⁻¹³M and the cells incubated at 37° C.,5% CO₂. AlamarBlue (Biosource) is added to all wells after 66 hours ofculture and the cells incubated further for 6 hours. If cells aregrowing, the alamarBlue is reduced and the colour of the media changesfrom blue to red. Plates are then read on a Fluostar (bmg) at 550 nm(excitation) and 590 nm (emission) and analysed by Prism (GrafPadsoftware).

A ref to Baf-3 cells:

Palacios, R. & Steinmetz, M. (1985) Cell 41 pp 727-734.

Description of a PEG-hIL-21 PK Study in Mice

The present experiment is to administer a single dose of PEG20K-hIL-21,PEG40K-hIL-21 and hIL-21 intravenously and subcutaneously to mice inorder to obtain bioavailability and pharmacokinetics characteristics ofPEG-hIL-21.

Material and Methods

Forty eight female C57BL/6Jbom weighing approximately 25 g fromBomholtgard, Ry, Denmark are included in the experiment.

During the study the animals will be kept and handled according tonormal procedure in the animal unit (Standard Operating Procedure no.010364) and are allowed free access to feed and water.

Test Formulations

hIL-21, PEG20k-hIL-21 and PEG40k-hIL-21 at a concentration of 200 μg/ml.The test substances will be dissolved in PBS buffer pH 7.4.

Dosing

The test substance will be dosed according to the following:

20 μg/25 g mouse corresponding to 0.8 μg/g mouse weight.

The i.v. injections will be given in the tail vein in a volume of 0.1ml.

The s.c. injections will be given on the back of neck in a volume of 0.1ml.

Blood Samples

Blood samples will be collected according to the following schedule:

After Intravenous Injection:

Predose, 5, 10, 20, 30, 45 (minutes), 1, 1.5, 2, 4 and 6 hours afterdosing.

After Subcutaneous Injection:

Predose, 10, 30 (minutes), 1, 1.5, 2, 3, 4, 6, 8 and 24 hours afterdosing.

Blood samples will be drawn from the orbital venous plexus.Approximately 0.1-0.2 ml blood will be drawn at each sampling time.Three blood samples will be taken from each animal. Blood samples fromtwo mice will be drawn at each time point.

Blood samples will be collected in Micronic test tubes and kept on icefor max 20 min before centrifugation (1200 m×g, 4° C., 10 min).

25 μl plasma sample will be transferred to Micronic tubes immediatelyafter centrifugation and stored at −20° C. until analysis.

Assay

The plasma samples will be analysed for the content of hIL-21 by aspecific immunoassay by Immunochemistry, Novo Nordisk A/S.

Plasma concentration-time profiles will be analysed by noncompartmentaland compartmental pharmacokinetic methods.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims and/or aspects appended hereto as permittedby applicable law.

1. A derivative of interleukin 21 (IL-21) or an IL-21 variant comprisingat least one polymeric molecule or lipophilic substituent.
 2. Thederivative of claim 1, wherein the polymeric molecule is one or more PEGgroups.
 3. The derivative of claim 2, wherein the polymeric molecule isone or more PEG groups having a weight of at least about 5 kDa.
 4. Thederivative of claim 3, wherein the derivative comprises one or more PEGgroups having a weight of at least about 30 kDa.
 5. The derivative ofclaim 1, wherein the polymeric molecule or lipophilic derivative isconjugated to the amino acid sequence of the IL-21 or IL-21 variant atthe C-terminus.
 6. The derivative of claim 1, wherein the polymericmolecule or lipophilic derivative is conjugated to the interior of theamino acid sequence of the IL-21 or IL-21 variant.
 7. The derivative ofclaim 1, wherein the polymeric molecule or lipophilic derivative isconjugated to the amino acid sequence of the IL-21 or IL-21 variant atthe N-terminus.
 8. The derivative of claim 1, wherein the IL-21 or IL-21variant is an IL-21.
 9. The derivative of claim 1, wherein the IL-21 orIL-21 variant is an IL-21 variant.
 10. The derivative of claim 9,wherein the polymeric molecule or lipophilic derivative is conjugated tothe IL-21 variant at an amino acid that does not naturally occur inhuman IL-21.
 11. A variant of interleukin 21 (IL-21) comprising anN-terminal addition of a sequence of 1-10 amino acids comprising a Ser,Tyr, Lys, or Cys residue.
 12. A variant of human interleukin 21 (hIL-21)comprising an addition of a serine residue at the N-terminus of thehuman IL-21 amino acid sequence (Ser-hIL-21).
 13. The IL-21 variant ofclaim 12, wherein the IL-21 variant is derivatized by conjugation of apolymeric group or lipophilic substituent to the N-terminal Ser residueof hIL-21.
 14. An isolated DNA comprising a sequence that codes for theexpression of Ser-hIL-21.
 15. A method of producing an IL-21 variantderivative comprising derivatizing Ser-hIL-21 with a polymeric moleculeunder conditions suitable for producing a derivative of Ser-hIL-21. 16.A method of treating cancer comprising administering to a patient inneed thereof an effective amount of a derivative according to claim 13.17. A method of treating infection comprising administering to a patientin need thereof an effective amount of a derivative according to claim13.